Os estudos abordando a regeneração dos tecidos dentários ganharam uma nova perspectiva com a utilização das células-tronco. E novas perspectivas têm surgido com a bioengenharia tecidual e as terapias periodontais e pulpares regeneradoras. O objetivo deste trabalho foi desenvolver o modelo experimental de autotransplante em ratos visando compará-lo à técnica de reimplante e estudar a capacidade terapêutica das células da medula óssea em diferentes biomateriais utilizados como matriz para a terapia de células-tronco no reparo dos tecidos dentais. Foram utilizados 23 ratos Wistar divididos em grupos de 1, 3, 15 e 60 dias para as técnicas de reimplante e autotransplante. Os grupos com injeção de células-tronco (CT) foram: (1) grupo de 3 dias, combinado à técnica de reimplante; (2) grupo de 15 dias com ambas as técnicas. Blocos contendo os três dentes molares superiores de cada lado dos ratos foram removidos, feitas radiografias periapicais e as peças foram processadas para inclusão em parafina. Foram avaliadas a espessura do ligamento periodontal (LPD) comparada entre os diferentes grupos e a morfologia celular e matriz extracelular relacionadas à superfície radicular, ao osso alveolar e à porção média do LPD, além das células da polpa dental de cada grupo. As células isoladas a partir da medula-óssea foram incubadas por 24h, 48h, e 72h em placas de cultura contendo membranas de colágeno bovino tipo I - CollaTape (Integra LifeSciences Corporation, Plainsboro, NJ, USA), enxerto ósseo - Extra Graft XG-13 (Silvestre Labs Quimica e Farmaceutica LTDA, RJ, Brazil) ou um dente molar de rato. Os espécimes foram observados em um microscópio invertido para contagem de células e processadas para observação no microscópio eletrônico de varredura (MEV). Os grupos de 1 e 3 dias apresentaram medidas de LPD significativamente maiores para a técnica de autotransplante quando comparadas ao reimplante. O grupo de 3 dias com CT não apresentou alterações pulpares significativas, diferente do controle (sem CT) O grupo de 15 dias com CT apresentou as mesmas características histológicas do grupo sem injeção de CT. A observação ao MEV dos biomateriais revelou que as células apresentaram pouca adesão e proliferação no enxerto ósseo e no cemento dentário quando comparados à membrana colágena. A técnica de reimplante associada à injeção de células-tronco sugere alguma influência da terapia com as células-tronco sobre a polpa. As distâncias aumentadas no LPD com a técnica de autotransplante podem não influenciar tanto o sucesso da técnica. As células mesenquimais da medula óssea possuem grande potencial para colonizarem a membrana colágena CollaTape que mostrou vantagens sobre o enxerto ósseo Extra Graft XG-13 como biomaterial para a aderência e a proliferação de células mononucleares da medula óssea, permitindo a diferenciação destas células. / The studies on the regeneration of dental tissues have gained a new perspective with the use of stem cells. And new perspectives have appeared with bioengineering and pulp and periodontal regenerative therapies. The aim of this study was to develop an experimental model of autotransplantation in rats in order to compare it with tooth reimplant technique and to study the therapeutic potential of bone marrow cells in different biomaterials used as scaffolds for stem cell therapy to repair dental tissues. 23 rats divided in 1, 3, 15 and 60 days groups were used for the techniques of tooth reimplant and autotranplant. The groups with stem cell injection (CT) were: (1) 3 days, combined with tooth replant technique; (2) 15 days with both techniques. Blocks containing the three molar teeth from each side of the rats superior jaws were removed, periapical radiographs were taken and the specimens were processed and embedded in paraffin. LPD thickness among different groups and cell morphology and extracellular matrix related to the root surface, alveolar bone and the middle portion of the LPD, and dental pulp cells were evaluated and compared from each group. Cells isolated from bone marrow were incubated for 24h, 48h and 72h in culture plates containing membranes of bovine collagen type I - CollaTape (Integra LifeSciences Corporation, Plainsboro, NJ, USA), bone graft - Extra Graft XG-13 (Silvestre Labs Chemical and Pharmaceutical Ltda, RJ, Brazil) or a mouse molar tooth. The specimens were observed using an inverted microscope for cell count and processed for observation in a scanning electron microscope (SEM). Groups 1 and 3 days showed LPD thickness significantly higher for tooth autotransplant technique when compared to reimplant. The group of 3 days with stem cells showed no significant pulp changes different from control (without stem cells). Group of 15 days with stem cells showed the same histological characteristics of the group without injection of stem cells. The biomaterials observation at SEM revealed that cells in the bone graft and in tooth cementum showed poor adhesion and prolifetarion when compared to collagen membrane The tooth reimplant technique associated with injection of stem cells suggests some influence of stem cells therapy on the pulp. The increased distances in LPD with tooth autotranplant technique may not influence the success of the technique. The bone marrow mesenchymal cells have great potential to colonize the collagen membrane CollaTape that showed advantages over bone graft Extra Graft XG-13 as a biomaterial for adhesion and proliferation of bone marrow mononuclear cells, allowing the differentiation of these cells.

La technique des membranes induites par un conformateur en polymethylmethacrylate (PMMA) et greffe d’os spongieux autologue a été décrite par Masquelet pour la régénération des pertes de substance osseuse segmentaire des os longs. Cette technique en deux temps pourrait avantageusement être utilisée pour la reconstruction des pertes de substances osseuses segmentaires mandibulaires en cancérologie en cas d’échec ou de contre-indication des greffes osseuse revascularisées. Le premier objectif de ce travail était d’évaluer les propriétés histologiques et biologiques des membranes induites par un conformateur en PMMA avec radiothérapie, et de les comparer avec celles induites par un conformateur en silicone. Ce matériau plus souple que le PMMA a été choisi comme alternative au PMMA dans le but de faciliter l’ablation du conformateur. Les membranes induites par le PMMA ou le silicone en sous-cutané chez des rats Wistar avaient une structure histologique et des propriétés biologiques comparables mais les résultats étaient plus stables pour les membranes induites par le silicone. Le second objectif de ce travail était de proposer un procédé d’ingénierie tissulaire en alternative à la greffe d’os spongieux autologue. La néoformation osseuse au sein d’une membrane induite par un conformateur en silicone avec greffe de différentes combinaisons de céramique phosphocalcique biphasique macroporeuse (MBCP+™), de moelle osseuse totale, de simvastatine et de rhBMP-2 a été évaluée chez des rats Wistar en sous-cutané puis en site osseux fémoral. Les résultats n’ont pas permis de montrer de néoformation osseuse significative dans les groupes avec simvastatine. Par contre, une néoformation osseuse significative a été montrée dans les groupes avec rhBMP-2, avec ou sans radiothérapie. Un effet substantiel de l’adjonction de moelle osseuse totale a également été retrouvé. / Induced membrane technique with a polymethylmethacrylate (PMMA) spacer and autologous cancellous bone graft has been proposed by doctor Masquelet for segmental long bone reconstruction. This two stage technique could be proposed for segmental mandibular bone reconstruction in oncological situations in case of failure or contraindication of revascularized autologous bone graft. The first objective of this study was to evaluate the histological and biological properties of membranes induced by PMMA with radiotherapy, and to compare them to membranes induced by silicone. This material smoother than PMMA has been chosen to facilitate spacer removal. Membranes induced by both materials in subcutaneous models in rats had similar histological and biological properties, but membranes induced by silicone were less affected by radiotherapy. The second objective of this study was to propose a tissue engineering procedure as an alternative to autologous bone graft. The new bone formation inside silicone induced membranes has been analyzed after implantation of different combinations of macroporous biphasic phosphocalcic ceramic (MBCP+™), total bone marrow, simvastatine and rhBMP-2 in subcutaneous and femoral osseous models in rats. No significant new bone formation has been demonstrated in simvastatin groups. However, a significant new bone formation has been demonstrated in rhBMP-2 groups, with or without radiotherapy. An increased new bone formation has also been demonstrated with total bone marrow.

A Síndrome do Intestino Curto (SIC) é uma doença complexa que ocorre após extensa ressecção do intestino delgado, levando a uma má absorção de nutrientes e fluidos, uma condição que pode causar diarreia, desnutrição e perda de peso graves com alto risco para o desenvolvimento da osteoporose. Estudos recentes mostram existir ampla interação fisiológica do esqueleto com os diversos sistemas, incluindo o metabolismo energético e o trato digestório. Peptídeos originados não só no tecido adiposo, mas também no intestino como as incretinas [GIP (polipeptídeo trópico insulínico dependente de glicose) e GLP1 (peptídeo 1 tipo glucagon)] modulam a atividade de remodelação óssea. O objetivo principal do atual estudo foi avaliar a relação entre os tecidos adiposos subcutâneo (TAS), visceral (TAV), lipídeos intra-hepáticos (LIH), tecido adiposo da medula óssea (TAMO), bem como do GIP, GLP1, e grelina com a densidade mineral óssea (DMO) em pacientes com SIC. Tratase de um estudo observacional prospectivo composto por dois grupos experimentais pareados por altura, idade e sexo: a) o grupo controle (GC) (n = 18; 9M,9F) e b) o grupo de pacientes com SIC, o qual foi avaliado em 2 ocasiões, com intervalo de um ano entre as análises, sendo denominados SIC0 (n = 14; 7M,7F) e SIC1 (n = 11; 6M,5F). Em comparação com o GC, pacientes com SIC ao longo do estudo apresentaram menor DMO e maior LIH e GIP (p< 0,05). Os valores de TAMO, GLP1 e grelina foram similares entre os grupos. O TAMO teve correlação negativa e significativa com DMO de L3 no GC (r= -0,6; p< 0,05), porém, no grupo SIC esta correlação foi positiva, mas sem significância estatística ao longo do estudo: SIC0 (r= 0,45; p= 0,13) e SIC1 (r= 0,45; p= 0,17). LIH associou-se negativamente com DMO do colo do fêmur (R²= 0,16; p< 0,05) e quadril total (R²= 0,27; p< 0,05). Existe alta prevalência de osteoporose em pacientes com SIC. No entanto, não se observou nem expansão de TAMO e nem relação negativa da DMO com o TAMO. O acesso a calorias parece afetar positivamente a relação entre TAMO e massa óssea. A deposição hepática de lipídeos parece afetar negativamente a massa óssea de pacientes com SIC. / Short bowel syndrome (SBS) is a complex disease that occurs after extensive resection of the small intestine leading to malabsorption of nutrients and fluids, a condition that can cause severe watery diarrhea, dehydration and acute weight loss, developing high risk for the appearance of osteometabolic disease. Studies have shown the progress on the physiological interaction of the skeleton with the various systems, including energetic metabolism and the gastrointestinal tract. Peptides originated not only in adipose tissue but also in the intestine such as incretin [GIP (Glucose-dependent insulinotropic polypeptide) and GLP1 (glucagonlike peptide 1) modulate bone remodeling activity. The main objective of the current study was to evaluate the influence of subcutaneous (SAT), visceral (VAT) adipose tissue, intrahepatic lipids (IHL), bone marrow fat adipose tissue (MAT), as well as the influence of GIP, GLP1, and ghrelin on the bone mineral density (BMD) of SBS patients. It is a prospective observational study composed by two experimental groups matched by height, age and sex: a) the control group (CG) (n = 18; 9M,9F) and b) the SBS group which were evaluated in two occasions with a period between analyzes of one year: named SBS0 (n = 14; 7M,7F) and SBS1 (n = 11; 6M,5F). Compared to CG, SBS patients throughout the study had significantly lower BMD and elevated IHL and GIP (p< 0.05). Values of MAT, GLP1 and ghrelin were similar between groups. MAT was negatively and significantly correlated with L3 BMD in the CG (r = -0.6; p< 0.05) and positively correlated, but not significant with L3 BMD in the SBS group throughout the study: SBS0 (r= 0.45; p= 0.13) and SBS1 (r= 0.45; p= 0.17). IHL was negatively and significantly associated with femoral neck BMD (R²= 0.16; p< 0.05) and total hip BMD (R²= 0.27; p< 0.05). The occurrence of osteoporosis is frequent in SBS patients, but MAT is not increased in these patients and had positive correlation with BMD, although not significant. Access to calories seems to positively affect the relationship between MAT and bone mass. IHL appear to negatively affect bone mass in SBS patients.

Transplante de medula óssea (TMO) é um procedimento terapêutico que tem como o objetivo a substituição da medula óssea doente por outra saudável. É utilizado em pacientes oncológicos e pode representar a cura da malignidade hematológica ou o resgate da medula óssea para a continuidade do tratamento antineoplásico. No TMO são empregadas drogas com alta toxicidade que agem ao nível sistêmico e que podem causar severos danos ao organismo. Esses danos resultam em complicações diversas que irão influenciar o prognóstico e sobrevida do paciente. As complicações pulmonares são associadas a altas taxas de mortalidade, sobretudo quando a ventilação mecânica (VM) é necessária. Ainda não há consenso na literatura quanto às causas, os fatores de risco e o tratamento adequado. Este estudo teve como objetivo identificar os fatores preditivos para insuficiência respiratória (IRP) em paciente oncológicos após o TMO autólogo e investigar o impacto da ventilação não-invasiva (VNI) na evolução clínica destes pacientes. Foi realizado o levantamento retrospectivo de 161 pacientes submetidos ao TMO autólogo no Hospital A.C. Camargo entre 1995 e 2005. Houve forte associação de IRP e óbito (p< 0,001) e também observamos associação com mucosite (p=0,016) e etilismo (p=0,036), essa associação permaneceu significante na análise multivariada [mucosite (p=0,004) e etilismo (p=0,02)]. De acordo com a análise de sobrevida encontramos associação com o maior número de regimes de quimioterapia no passado (p= 0.005), mucosite (p= 0.029), etilismo (p= 0.044) e redução da capacidade de difusão de monóxido de carbono (p=0.048). Em nosso estudo a taxa de mortalidade permanece alta para aqueles pacientes que desenvolvem IRP e necessitam de VM. A VNI não demonstrou efeito protetor na sobrevida dos pacientes que evoluíram com IRP. A mucosite mostrou-se um fator de piora no prognóstico destes pacientes devendo ser agressivamente evitada e tratada. O impacto do etilismo na incidência de insuficiência respiratória (IRP) e mortalidade destes pacientes merece destaque especial com necessidade de mais pesquisas. O stress oxidativo parece ter um importante efeito causal para as complicações pulmonares após o TMO podendo ser potencializado pelo etilismo. O maior número de esquemas de quimioterapia no passado aumentou a mortalidade, isso poderia representar pacientes com neoplasias mais resistentes ao tratamento ou pacientes que foram expostos ao efeito cumulativo das drogas. A capacidade de difusão de monóxido de carbono é um teste bastante útil para prever risco de óbito / Bone marrow transplantation (BMT) is a therapeutic procedure to replace unable marrow for another healthy one. Its used in cancer patients to cure or refresh marrow to keep the cancer treatment. Respiratory failure (RF) after BMT is associated with high mortality specially when mechanical ventilation (MV) is needed, it may be due to treatment-related toxicity, infection, or immunologic insufficiency. Many studies have trying to identify causes, predicting factors and response for the usual treatment, but until now there is no agreement in literature. The aim of this study is to identify which factors evaluated in routine anamnesis and exams pretransplant can affect the prognosis of those patients. We retrospectively collected variables in 161 consecutive cancer patients who had undergone autologous BMT. The variables obtained from the in-hospital period were submitted to univariated and multivariated stepwise logistic regression analyses. Survival analysis also was computed in 100 days follow up. There were highest association for respiratory failure (RF) with death (p<0.001) and we also found a significant association with alcohol abuse (p =0.036) and mucositis (p=0.016), and those variables remained statically significant in multivariated analysis [mucositis (p=0.004) and alcohol abuse (p=0.02)]. According to survival analysis we found significance for the major number of chemotherapy regimens received in the past (p= 0.005), mucositis (p= 0.029), alcohol abuse (p= 0.044) and decreased monoxide carbon diffusion (p=0.048). In our study the mortality rate remains high for those patients who develop RF and need MV. It seems not to have impact what kind of ventilatory support is used (invasive or non-invasive ventilation). Mucositis needs special attention because treating it we can be preventing RF and decrease mortality rates. The effect of alcohol abuse in mortality rate and RF deserve a special attention because its socially accepted and his deleterious action its not explained. The oxidative stress seems to have an important main effect over post-transplant complications and it can be increased by alcohol abuse history. The major number of chemotherapy regimens received in the past increase mortality, it could represent patients who had baseline disease more difficult to treat, more resistant, or patients who were exposed to a cumulative side effect of drugs. Monoxide carbon diffusion is a useful test to identify the risk for death

Introdução: Em câncer de mama, existem evidências de que o microambiente pode influenciar o desenvolvimento do tumor no sítio primário, bem como em metástases regionais e a distância. Neste contexto, fibroblastos são importantes células estromais que podem influenciar a proliferação e a migração de células do câncer e podem prover um nicho apropriado para o desenvolvimento tumoral. Objetivos:O principal objetivo deste trabalho é comparar células estromais obtidas do tumor primário (PT), metástase linfonodal (N+) e medula óssea (BM) de pacientes com câncer de mama, através do perfil de expressão gênica. Pacientes e Métodos: Foi analisada a expressão gênica de fibroblastos (cultura primária) de 11 pacientes com câncer de mama. O perfil de expressão foi determinado em PT (n=4), N+(n=3) e BM (n=4) através de uma plataforma de cDNA microarray customizada (contendo 4.800 sequencias imobilizadas, representando cerca de 4600 genes), e os genes diferencialmente expressos foram identificados pelo teste SAM multiclasse, seguido pelo teste SAM de duas classes (TMEV, FDR 0%). A análise funcional foi realizada pelo software DAVID v6.7. Validação técnica foi realizada em 6 amostras previamente analisadas no microarray e a validação biológica em fibroblastos obtidos de outros 16 pacientes utilizando-se de RT-qPCR. Resultados: O perfil de expressão gênica dos fibroblastos obtidos de diferentes sítios mostraram 267 genes diferencialmente expressos, os quais apropriadamente agruparam os fibroblastos de acordo com suas origens (PT vs. N+ vs. BM). Apesar das diferenças entre PT e N+ serem representadas por 20 genes, as diferenças entre PT vs. BM e N+ vs BM foram mais significantes (235 e 245 genes diferencialmente expressos respectivamente). Análise funcional dos genes diferencialmente expressos mostrou enriquecimento de funções relacionadas ao desenvolvimento e morfogênese.A seguir, a expressão de alguns genes selecionados foi analisada em uma série diferente de amostras (validação biológica). Desse modo observamos que NOTCH2 confirmou uma alta expressão em N+ (vs. PT), e ADCY2, HECTD1, HNMT, LOX, MACF1 e USP16 confirmaram alta expressão em BM (vs PT). Conclusão:Em pacientes com câncer de mama, células estromais obtidas de diferentes origens apresentam um perfil de expressão gênica diferencial, o qual pode influenciar o comportamento do tumor / may influence tumor development in the primary site of breast cancer, as well as in regional and distant metastatic sites. In this context, fibroblasts are important stromal cells which influence proliferation and migration of cancer cells and may also provide an appropriate niche to tumor development. Objectives: The main objective of this work is the comparison of stromal cells from the primary tumor (PT), lymph node metastasis (N+) and bone marrow (BM) obtained from breast cancer patients, through gene expression profile. Patients and Methods: The gene expression profile was analyzed in fibroblasts primary culture from 11 breast cancer patients. The expression profiles of PT cells (n=4), N+ cells (n=3) and BM cells (n=4) were determined through a customized cDNA microarray platform (containing 4800 immobilized sequences which represents 4600 genes approximately). The analysis were performed by SAM multiclass (TMEV; FDR 0%), followed by SAM two classes test (TMEV; FDR 0%). Functional analysis was performed using DAVID v6.7. Technical validation was performed in same 6 samples that were previously analyzed in microarray experiments and biological validation was performed in fibroblasts obtained from other group of 16patients by RT-qPCR Results: The expression profile of fibroblasts obtained from three sites revealed 267 differentially expressed genes, which appropriately clustered fibroblasts in three different branches, in accordance with their origin (PT vs. N+ vs. BM). Although the differences between PT and N+ were represented by 20 genes, differences between PT vs. BM and N+ vs. BM were more significant (235 and 245 differentially expressed genes respectively). Functional analysis revealed enrichment of functions related to development and morphogenesis. Afterwards, the expression of some selected genes were analyzed in a different batch of samples (biological validation).Thereby, NOTCH2 confirmed high expression in N+ (vs. PT), and ADCY2, HECTD1, HNMT, LOX, MACF1 and USP16 confirmed high expression in BM (vs. PT). Conclusion: In breast cancer patients, stromal cells obtained from different origins present a differential gene expression profile, which may influence tumor behavior

Chez l’adulte les premières étapes du développement hématopoïétique se déroulent dans la moelle osseuse (MO). La contribution de cellules d’origine mésenchymateuse, appelées niches stromales, a été démontrée dans le cas de la maintenance des cellules souches hématopoïétiques (CSH) et du développement des lymphocytes B (LB). Ainsi la maintenance des CSH dépend de niches périvasculaires sécrétant CXCL12 et SCF. Par ailleurs les LB les plus précoces (preproB) sont en contact de cellules stromales CXCL12+, puis migrent vers des cellules stromales exprimant l’interleukine-7 lors de leur différentiation en cellules proB. L’expression du préBCR, marque ensuite l’entrée dans le stade préB. À ce stade, les cellules sont au contact de cellules stromales galectine-1+.Malgré les progrès obtenus dans la compréhension du rôle des niches stromales, leur hétérogénéité et les mécanismes contrôlant la migration et l’adhésion des cellules hématopoïétiques en différenciation restent à mieux définir. Dans cet objectif, nous avons caractérisé phénotypiquement les cellules stromales de la MO mais aussi démontré l’existence d’une niche multi-spécifique, associée aux sinusoïdes et capable de soutenir les CSH et les LB.La contribution des niches dans le développement et la résistance aux traitements des Leucémies Aigues Lymphoblastiques de type B (LAL-B), équivalents pathologiques des LB en développement, a aussi été démontrée. Au cours de mon travail de thèse nous avons révélé l'influence d'un facteur exprimé par des cellules stromales de la MO sur la prolifération des LAL-B. À terme, ces travaux permettront de développer des traitements ciblant les fonctions protectrices des niches tumorales. / In adults, the early stages of hematopoietic development take place in the bone marrow (BM). The contribution of specialized cells of mesenchymal origin, called stromal niches, has been demonstrated in the case of hematopoietic stem cell (HSC) maintenance and B lymphocyte development. Indeed, the maintenance of HSC depends on perivascular niches secreting CXCL12 and SCF. Furthermore progenitor B cells (preproB) are in contact with CXCL12+ stromal cells and migrate towards interleukin 7 expressing stromal cells during their differentiation into proB cells. PreBCR expression then marks the entrance into the preB cell stage. At this point, the cells are in contact with galectin-1+ stromal cells.Although progress have been made in understanding the role of stromal cell niches, their heterogeneity and the mechanisms controlling migration and adhesion of differentiating hematopoietic cells are controversial and remain to be defined. With this objective, we characterized phenotypically BM stromal cells but also demonstrated the existence of a multi-specific niche, associated to sinusoids and able to support both HSC and early B cells.The contribution of BM niches in the development and resistance to treatment of B cell Acute Lymphoblastic Leukemia (B-ALL), pathological equivalent of developing B cells has also been demonstrated. During my PhD, our work revealed the influence of a factor expressed by BM stromal cells on the proliferation of B-ALL. Ultimately, this work will allow the development of treatments targeting the protective functions of tumor niches.

Ziele der hier vorliegenden Arbeit waren eine immunhistochemische Analyse von GFAP (‚glial fibrillary acidic protein’) und GLUT-1 (Glukosetransporter-1) nach fokaler zerebraler Ischämie sowie deren mögliche Beeinflussung durch eine intravenöse Transplantation autologer mononukleärer Knochenmarkzellen (mKMZ) im Schafmodell. Eine differenzierte Analyse der Zielstrukturen in grauer und weißer Substanz (GS bzw. WS) sollte Aufschluss über eventuell unterschiedliche Reaktionsmuster liefern. Das Gehirnmaterial von zehn Tieren der bereits 2006/2007 stattgefundenen Studie, welche mit PET und MRT-Untersuchungen sowie der Durchführung von Verhaltenstests einherging, wurde retrospektiv im Rahmen der vorliegenden Arbeit untersucht. Je fünf gehörten zu einer Kontroll- bzw. Therapiegruppe (KG bzw. TG). Bei allen Versuchstieren wurde durch die permanente Okklusion der linken mittleren Zerebralarterie (pMCAO) eine fokale zerebrale Ischämie im Bereich des Neokortex hervorgerufen. Die Tiere der Therapiegruppe erhielten 24 Stunden nach dem Eingriff eine Transplantation autologer mKMZ (4x106/kg KGew). Nach sieben Wochen wurden die Versuchstiere getötet, ihre Schädel perfundiert und ihre Gehirne fixiert. Eine Lamelle der Gehirne wurde für die anschließende histologische Untersuchung in 30% Saccharose konserviert. Nach der Etablierung der Antikörper GFAP und GLUT-1 wurden vier Regionen der Gehirn-lamellen immunhistochemisch markiert und abschließend qualitativ und quantitativ analysiert. Die Regionen I (infarktnah) und III (infarktfern) lagen in der ipsilateralen Hemisphäre, die Regionen II (korrespondierend zu Region I) und IV (korrespondierend zu Region III) in der kontralateralen Hemisphäre. Durch den höheren Substanzverlust an Gehirnmasse in der ipsi-lateralen Hemisphäre der KG, wurden in dieser Tiergruppe die Regionen III und IV nicht ausgewertet. Vor der Analyse sind die physiologischen Markierungsmuster der vier Regionen in grauer und weißer Substanz an zwei gesunden Tieren (Prozesskontrolle) aufgezeigt worden. Durch die elektronenmikroskopische Untersuchung von Präparaten und anhand von GFAP/GLUT-1 doppelmarkierten Präparaten konnte festgestellt werden, dass die Astrozytenendfüßchen durch den hier verwendeten GLUT-1 Antikörper nicht markiert wur-den, sondern dass alleinig die gefäßständige, 55 kDa schwere Isoform detektiert worden ist. Die fokale zerebrale Ischämie führte in beiden Gruppen zu einer hochgradigen reaktiven Astrogliose mit Ausprägung einer Glianarbe in Region I. Protoplasmatische Astrozyten der grauen und fibrilläre Astrozyten der weißen Substanz zeigten hypertrophe Veränderungen. Die reaktive Astrogliose von Region I spiegelte sich in einer erhöhten GFAP-Dichte wider (p<0,05 in der Therapiegruppe). Region III hatte die gleiche GFAP-Dichte wie die Regionen II und IV. Der direkte Vergleich zwischen den Regionen I der beiden Gruppen zeigte Veränderungen der GFAP-Dichte durch die Zelltherapie auf: In der GS der Therapiegruppe lag eine geringere GFAP-Dichte vor, in der WS eine höhere (≠ p<0,05; GS und WS). Die Ergebnisse der GLUT-1-Analyse sind denen der GFAP-Analyse sehr ähnlich. Durch den Schlaganfall ist es zu einer erhöhten GLUT-1-Expression in GS und WS (p<0,05 WS) von Region I der Kontrollgruppe gekommen. Auch in Region I der Therapiegruppe konnten er-höhte GLUT-1-Dichten in GS und WS (p<0,05 WS) detektiert werden, zusätzlich dazu lag in der GS von Region III der Therapiegruppe eine erhöhte GLUT-1-Dichte vor (p<0,05). Der Vergleich zwischen beiden Gruppen zeigte Veränderungen durch die Therapie für die Regio-nen I und II auf. Die GLUT-1-Dichte der WS war in beiden Regionen in der TG erhöht (p<0,05), die GS von Region I zeigte in der Therapiegruppe eine geringere GLUT-1-Dichte. Ein Schlaganfall führt zu einer Erhöhung der GFAP sowie GLUT-1-Dichten in WS und GS im infarktnahen Gebiet. Durch die Transplantation von 4x106 autologen mononukleären Knochenmarkzellen pro kg KGew 24 Stunden nach dem Schlaganfall können diese Strukturen in ihren Expressionsmustern beeinflusst werden, dabei reagieren graue und weiße Substanz unterschiedlich: Die GS mit einer Verringerung, die WS mit einer Erhöhung der GFAP- bzw. GLUT-1-Dichte (p<0,05 WS, GLUT-1). Die Funktionskreisläufe in infarktfernen Regionen sind sieben Wochen nach dem Schlaganfall auf Astrozytenebene normalisiert (vgl. Region III). Die erhöhte GLUT-1-Dichte (p<0,05) in der GS der infarktfernen Region ist möglicherweise mit einem erhöhten Glukosemetabolismus in Verbindung zu setzen. Dies kann jedoch erst durch die Auswertung der FDG-PET-Daten beantwortet werden. Ob die durch Transplantation autologer mKMZ festgestellten Veränderungen der GFAP- und GLUT-1-Dichte in der Therapiegruppe zusätzlich mit einer verbesserten motorischen Leistung der Tiere einhergingen, wird erst durch die Analyse der Daten aus den Verhaltenstests festgestellt werden können.

Dendritic cells (DCs), a hematopoietic cell type belonging to the sub-group of cells called antigen presenting cells (APCs), inhabit a central role in innate and adaptive immunity. Although the DC family is very heterogeneous, all members share unique features. Most importantly, DCs can stimulate an immune response. This is due to the cells’ ability to capture and process antigens and to maturate in the presence of danger signals presented by pathogens. Maturation in turn results in the migration of DCs from the tissue they reside in to the draining lymph nodes, as well as in the subsequent presentation of the acquired antigens to T cells. In the skin, which is one of the most immunogeneic organs, DCs are present in sizable numbers in both the epidermis and the dermis. This study focused on two types of DCs: epidermal Langerhans cells (LCs) and dermal DCs (DDCs). While much is understood about LCs, far less is known about the role that DDCs play in skin immunity. Therefore one purpose of this study was to characterize DDCs and to compare their phenotype and functions to that of LCs. This study used two different methods to characterize human skin resident immune cells with regard to their number and distribution. First, a stable analytical immunohistochemistry-based method was developed and applied to a substantial number of healthy skin donors. This enabled a quantitative analysis of skin DC types and skin resident T cells at different anatomical locations in situ. A novel method to count dermal cell populations in situ was developed that resulted in the first published quantification of APCs, DDCs, as well as T cells in human dermis. Second, the traditional form of the emigration assay, which selectively enriches vital cells capable of ex vivo emigration from the skin, was upgraded toward a stable analytical method to separate epidermal LCs from DDCs. In this way, both skin DC types became accessible in sufficient numbers to allow for a comparison of phenotypes and functions in vitro. The resulting phenotypic observations clearly showed that both, LCs and DDCs are not fully mature after their emigration ex vivo and that both can be transformed into a phenotypically more mature state by treating them with inflammatory cytokines. What’s more, LCs are also functionally in an immature state after their emigration. They efficiently took up antigen, showed a low capacity to trans-migrate in response to chemokines, and demonstrated a low capacity to stimulate allogeneic T cells in a mixed leukocyte reaction (MLR). For the first time this study observed all these main APC functions not only for LCs but additionally for DDCs. As these observations were made in relation to LCs of the same donor, it could be concluded that DDCs are functionally more mature than LCs after emigration. DDCs showed a lower antigen uptake capacity than LCs but were superior in terms of their migratory and stimulatory capacity. However, treatment with cytokines could skew LC functions toward functional capacities observed for DDCs, i.e., it decreased LCs’ Ag uptake and increased their migratory and stimulatory capacity, whereas the cytokine treatment did not alter DDCs’ functional capacities. After improving immuno-histochemistry and the emigration assay using healthy skin samples, these newly developed techniques were implemented in clinical trials to observe the number, distribution and migratory capacity of skin DCs and T cells in patients undergoing allogeneic hematopoietic cell transplantation (aHSCT). Such a study is of importance because the turnover of DCs and T cells is closely associated with the occurrence of acute graft-versus-host disease (aGvHD), the major cause of morbidity and mortality after aHSCT. Due to the study design used, this study concisely demonstrate that at the onset of aGvHD, different DC types accumulate along with effector T cells in skin lesions of aGvHD but not in uninvolved skin of the same patient. These results suggest that in addition to donor T cells LCs and DDCs play a role during the early phase of cutaneous aGvHD directly within the site of inflammation. The view of many authors that DC depletion in the transplant recipient, especially in target organs, is a promising approach for aGvHD prophylaxis and therapy is further underscored by these results. One targeting strategy to inhibit GvHD by eliminating recipient DCs may be the use of DC specific monoclonal antibodies. Alemtuzumab (anti-CD52) is a monoclonal antibody and has proven effective in preventing aGvHD after aHSCT. It may, despite depleting donor T cells, also work by targeting recipient DCs. To determine whether the last mechanism of action is significant, a second clinical study investigated the effects of intravenous alemtuzumab on DCs by comparing the number of these cells in skin and blood of patients before and after a 4-week course of alemtuzumab treatment. The result was that although skin DCs weakly express the target antigen CD52 the number of these cells was not consistently reduced by alemtuzumab. In contrast, circulating blood DCs have a stronger CD52 expression and were significantly reduced by the treatment. In conclusion, this work provides new insights into the phenotypical and functional characteristics of human skin DCs, as well as into the fate of these cell types during aHSCT. The investigation of the APC system during aGvHD as carried out here will help to understand the process of aGvHD in more detail. All these efforts may hopefully support the development of new approaches for therapy and prevention of this major limitation of aHSCT and may help to improve this only curative therapy for several life-threatening diseases.

No description available.

L’administration de fer dextran à 10 mg/kg intramusculaire (IM) est un traitement empirique couramment recommandé en médecine aviaire lors d’hémorragie ou d’anémie. L’objectif principal de cette étude était d’évaluer les effets de ce traitement sur l’anémie chez les oiseaux de proie. Deux types d’individus ont été utilisés : des crécerelles d’Amérique (Falco sparverius) où une anémie par perte de sang externe aiguë a été créée (deux phlébotomies de 20-40 % du volume sanguin total à un intervalle de 6 h) et des oiseaux de proie sauvages de différentes espèces souffrant d’anémies diverses. L’ensemble des oiseaux a été subdivisé aléatoirement en groupe traitement (fer dextran 10 mg/kg IM) et contrôle (NaCl 0,9% IM). Un suivi dans le temps a été réalisé afin d’étudier leur récupération de l’anémie, la présence d’effets secondaires au traitement et l’impact d’une administration de fer sur ces réserves. Aucune différence significative n’a été observée entre les deux groupes en ce qui concerne les signes cliniques, l’hématocrite, le pourcentage des polychromatophiles/réticulocytes, la densité cellulaire et le fer de la moelle osseuse, la créatine kinase et le fer plasmatique. La majorité des crécerelles ont présenté une myosite au site d’injection du fer. Nos résultats suggèrent qu’une administration de 10 mg/kg de fer dextran IM n’a pas d’effet sur l’érythropoïèse des rapaces souffrant d’anémie par perte de sang externe aiguë, qu’elle provoque une légère inflammation au site d’injection et qu’elle n’influence pas les réserves de fer. Le comptage des réticulocytes en anneau et des polychromatophiles semble être deux méthodes équivalentes. / A 10 mg/kg intramuscular (IM) administration of iron dextran is a common empirical treatment recommended in avian medicine for hemorrhage and anemia. The purpose of this study was to evaluate the effects of this treatment on anemia in birds of prey. Two kinds of specimen were used: the American kestrel (Falco sparverius) where an acute external blood loss anemia was created (with two phlebotomies of 20-40 % of the total blood volume at 6 hours interval) and other various species of wild birds of prey suffering from different types of anemia. All subjects were randomized into a treatment (iron dextran 10 mg/kg IM) or a control (NaCl 0,9 % IM) group. Monitoring was carried out to evaluate the evolution of the anemia, presence of side effects and impact of an iron administration on their iron reserve. No significant differences were observed between the two treatment groups for clinical signs, packed cell volume, the percentage of reticulocytes/polychromatophilic erythrocytes, bone marrow cellularity and iron, plasmatic iron and creatine kinase. Most kestrels had a myositis at the iron injection site. Our results suggest that an IM injection of 10 mg/kg iron dextran has no effect on raptor erythropoiesis after an acute external blood loss anemia, that it has no effect on iron reserve, and that it can cause mild inflammation at the injection site. The polychromatophilic erythrocytes and the reticulocytes ring form count were two equivalent methods.